Novel compounds are actively pursued as leads for drug discovery. Chemically, novel leads provide insight about the recognition determinants of the target receptor. Biologically, they can have specificities that substrate analogs lack and elude barriers or defenses to which substrate analogs fall victim.
The need for such new biological effects is keenly felt in the search for inhibitors of β-lactamases. These enzymes are the major resistance determinants to β-lactam antibiotics, including the penicillins and the cephalosporins, and threaten public health. To combat these enzymes, β-lactam inhibitors such as clavulanic acid, or “β-lactamase resistant” β-lactams such as ceftazidime, have been introduced (FIG. 1). The similarity of these β-lactams to the original substrates has allowed resistance to develop further. Broad-spectrum β-lactamases, such as the class C β-lactamase AmpC, have spread among bacteria. Point substitutions have resulted in mutants of once narrow-spectrum class A β-lactamases, leading to enzymes like TEM-30 and TEM-64 that are either less inhibited by, or can simply hydrolyze, the “β-lactamase resistant” compounds. Recently, new substrate analogs have been described that can inhibit these mutant and broad-spectrum β-lactamases with IC50 values as low as 100 nM (FIG. 1). Given their similarity to substrates, resistance rapidly develops against these new agents as well.
A more ambitious strategy abandons substrate information altogether, focusing instead on the structure of the receptor as the sole template for design. Structure-based screening approaches have discovered inhibitors dissimilar to both substrates and substrate analogs. These novel inhibitors may evade traditional, pre-evolved resistance mechanisms. Conversely, however, these novel inhibitors of AmpC β-lactamase are relatively weak, with K, values in the 25 μM range.
Between the extremes of substrate analogs and structure-based discovery lie transition-state analogs (FIG. 2), such as boronic acids. These inhibitors replace the β-lactam recognition motif with a boronic acid, which makes a reversible, dative covalent bond with the active site serine residue forming a tetrahedral adduct (FIG. 2a).
Replacing the lactam group with a boronic acid permits evasion of many of the resistance mechanisms that now jeopardize β-lactams. By deploying side chains normally found in lactam substrates, it has been possible to improve the potency of these compounds, down to 5.9 nM for TEM-1. Glycylboronic acids (See FIG. 2b and Table 1, cpds. 1-4) were previously found to inhibit AmpC competitively, with Ki values as low as 20 nM.
The glycylboronic acids resemble half of the β-lactam molecule, bearing the R1 side chain of substrates but lacking recognition elements corresponding to the thiazolidine or dihydrothiazine rings of penicillins or cephalosporins, respectively (FIG. 1a). The absence of a negatively charged group in a position corresponding to the C4′ position of dihydrothiazine ring seems particularly noteworthy. All β-lactams bear a carboxylic or sulfonic acid at this position. In class A β-lactamases, this group is a key recognition element. In class C β-lactamases the role of this group is less understood. In fact, mutant and substrate analyses of the prior art suggest that such a charged group is not needed for recognition.